www.nature.com/scientificreports OPEN ECM alterations in Fndc3a (Fibronectin Domain Containing Protein 3A) defcient zebrafsh Received: 22 April 2019 Accepted: 5 September 2019 cause temporal fn development Published: xx xx xxxx and regeneration defects Daniel Liedtke 1, Melanie Orth1, Michelle Meissler1, Sinje Geuer2,3, Sabine Knaup1, Isabell Köblitz1,4 & Eva Klopocki 1 Fin development and regeneration are complex biological processes that are highly relevant in teleost fsh. They share genetic factors, signaling pathways and cellular properties to coordinate formation of regularly shaped extremities. Especially correct tissue structure defned by extracellular matrix (ECM) formation is essential. Gene expression and protein localization studies demonstrated expression of fndc3a (fbronectin domain containing protein 3a) in both developing and regenerating caudal fns of zebrafsh (Danio rerio). We established a hypomorphic fndc3a mutant line (fndc3awue1/wue1) via CRISPR/ Cas9, exhibiting phenotypic malformations and changed gene expression patterns during early stages of median fn fold development. These developmental efects are mostly temporary, but result in a fraction of adults with permanent tail fn deformations. In addition, caudal fn regeneration in adult fndc3awue1/wue1 mutants is hampered by interference with actinotrichia formation and epidermal cell organization. Investigation of the ECM implies that loss of epidermal tissue structure is a common cause for both of the observed defects. Our results thereby provide a molecular link between these developmental processes and foreshadow Fndc3a as a novel temporal regulator of epidermal cell properties during extremity development and regeneration in zebrafsh. A wide number of conserved genetic and structural features have been identifed regulating fn development in ray fnned fsh species, like zebrafsh (Danio rerio), and imply shared mechanisms throughout evolution1. Te embryonic development of pectoral fns in fsh species is assumed to resemble limb development in higher verte- brates, with common molecular signals arising from a structure called the apical ectodermal ridge (AER)2,3. Only recently diferences between fn and limb AER have been reported and hint at a fn specifc cellular process3,4. Moreover, there are also eminent developmental diferences between paired fns (pectoral fns) and unpaired fns (caudal, anal and dorsal fns). All unpaired fns arise from a common developmental precursor structure, called the median fn fold (mf), which is exclusively found in teleosts5. An increasing number of molecular processes and distinct genes are still being identifed by forward and reverse genetic screens in zebrafsh, revealing a com- plex network of factors necessary for correct median fn fold development and function6,7. Already more than 30 years ago changes of epidermal cell shape and modulation of the extracellular matrix (ECM) have been described as one of the essential factors for correct median fn fold and caudal fn morphogenesis in zebrafsh8. Recently, the Wnt signaling pathway has been shown crucial for regulation of epithelial cell morphology by modulating laminin levels and thereby orchestrating correct ECM patterning in growing fns9. Tese early cellular steps of caudal fn development are prerequisites for subsequent processes; i.e. mesoderm cell migration, cell diferenti- ation, fn growth, development of cartilage and bone and the gradual resorption of the median fn fold during 1Institute of Human Genetics, Julius-Maximilians-University, Würzburg, Germany. 2Institute for Medical Genetics and Human Genetics, Charité Universitätsmedizin Berlin, Berlin, Germany. 3Center for Human Genetics, Bioscientia, Ingelheim, Germany. 4Department of Cell and Developmental Biology, Julius-Maximilians-University, Würzburg, Germany. Correspondence and requests for materials should be addressed to D.L. (email: [email protected] wuerzburg.de) SCIENTIFIC REPORTS | (2019) 9:13383 | https://doi.org/10.1038/s41598-019-50055-w 1 www.nature.com/scientificreports/ www.nature.com/scientificreports juvenile stages10. Particularly fn rays are essential structural elements formed by the assembly of actinotrichia and lepidotrichia at the basal membrane of epidermal cells11,12. While lepidotrichia are segmented and calcifed bone rays, actinotrichia are non-calcifed fbers with a characteristic brush-shaped structure. Actinotrichia fbers display a transverse striation which can be observed in several fsh species via electron microscopy suggesting these fbrils to be hyperpolymerized collagen13. Two collagens, Col2a1 and Col1a1, as well as actinodin proteins, encoded by and1, and2, and3 and and4, are essential for actinotrichia formation at the fn tips during fn devel- opment and regeneration14–16. It is well accepted that a number of conserved molecular mechanisms are shared between extremity devel- opment during embryogenesis and fn regeneration in adult fsh, a developmental process enabling complete replacement of lost tissues17,18. For both processes correct epidermal cell function, epithelial cell structure, and actinotrichia fber assembly are essential to correctly build all skeletal and mesenchymal fn elements14,19,20. Structural factors, especially ECM proteins like integrins and laminins, have been implied in regulating Wnt signaling during regeneration and in correct assembly of the teleost fn6,9,21. Although a large number of involved “molecular players” have been described to date, not all factors necessary for correct ECM assembly in the regen- erating caudal fn or correct median fn fold development and function have been elucidated yet. FNDC3A protein has initially been described to be overexpressed in human odontoblasts22 and consists of up to nine fbronectin type III domains, which are a common feature of a large number of extracellular pro- teins acting by modulation of diferent signaling pathways23,24. Functional experiments in Symplastic spermatids (sys) knockout mice indicated that FNDC3A is essential for cell adhesion between spermatids and Sertoli cells, resulting in sterile males25. Further developmental functions of FNDC3A in vertebrates are still unknown and an association to extremity development in mammals has only been described recently by the Mouse Organogenesis Cell Atlas, showing expression of Fndc3a in epithelial cells of the limb AER26. Te purpose of this study was to investigate potential functions of Fndc3a during vertebrate extremity deployment and regeneration in zebrafsh (Danio rerio). Results Phylogenetic and syntheny analyses showed that the FNDC3A gene is highly conserved throughout vertebrate evolution and orthologues are not duplicated in ray-fnned fsh species (data not shown). In the zebrafsh genome fndc3a is located on chromosome 15 and encodes in 29 exons for two diferent transcripts that are highly similar, with corresponding proteins of 1247 and 1217aa that only difer in a 30aa stretch at the N-terminus (ENSEMBL Zv9: 3,066,162-3,114,443 reverse strand; ENSDARG00000067569; ZFIN ID: ZDB-GENE-030131-7015; GenBank: XM_021466300.1, XM_021466301). Zebrafsh Fndc3a protein (UniProt: A0A140LGL5) consists of one transmembrane domain located at the C-terminus, 9 fbronectin type III domains and one signal pep- tide located at the N-terminus. Amino acid alignment resulted in an up to 57% amino acid identity with 95% coverage, indicating a high level of conservation between human and zebrafsh proteins. Furthermore, two fndc3a paralogues can be identifed in the zebrafsh genome: fndc3ba (chromosome 2; ENSDARG00000078179; ZFIN ID: ZDB-GENE-070510-1) and fndc3bb (chromosome 24; ENSDARG00000062023; ZFIN ID: ZDB-GENE-070510-2). Both genes share highest sequence similarities with FNDC3B and form a distinct sub- group aside from FNDC3A gens. Amino acid alignment comparison of both zebrafsh paralogous to human FNDC3B show for Fndc3ba up to 68%% amino acid identity by 98% coverage, while Fndc3bb shows up to 56% by 98% coverage. Both zebrafsh proteins show typical FNDC3 protein domain structure, by displaying one trans- membrane and 9 fbronectin type III domains. Syntheny analyses furthermore indicated the location of both genes within two distinct duplicated genomic regions on zebrafsh chromosomes 2 and 24. Both regions share up to 8 additional duplicated genes fanking zebrafsh fndc3b genes, which are also located within the human FNDC3B locus. All three gene family members have not been functionally investigated in zebrafsh yet. Expression of fndc3a during early zebrafsh development. Earliest expression of fndc3a can be detected via RT-PCR and RNA-seq during blastula stages and indicate maternal transcripts of fndc3a (data not shown). To resolve the spatiotemporal expression of fndc3a during zebrafsh development, we performed RNA in-situ hybridization experiments (Fig. 1). fndc3a transcripts were detected in a broad pattern and in number of diferent tissues, but showed cell type restricted expression within the tail bud region and the ventral median fn fold from 14 hpf onwards (hpf = hours post-fertilization; Fig. 1A,B; for visualization also of weak expression within the tailbud cells embryos shown in B are longer stained with NBT/BCIP). Expression in the tail bud region is changing during the next hours of development and could be detected apart from the median fn fold, in the cloaca, and in cells of the chordo neural hinge region (Fig. 1B). From 14 hpf onwards fndc3a was additionally present in distinct brain regions, the notochord, somites, pectoral fns and the